chemopreventive effect of omega 3 polyunsaturated fatty acids and atorvastatin in rats with bladder cancer

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Tumor Biology February 2017: 1 –12

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Introduction

Bladder cancer is one of the most common urinary system

malignancies worldwide occurring in both sexes It is

more frequent in men than women, and the incidence and

mortality rate of bladder cancer greatly increased in the

recent years.1 Cigarette smoking is the main risk factor for

the development of urothelial tumor Other risk factors

may include exposure to arsenic in drinking water,

occupa-tional exposure to aromatic amines, and genetic alterations

such as mutation in P53 gene.2,3

Omega-3 polyunsaturated fatty acids (n-3 PUFAs), espe-cially eicosapentaenoic acid (EPA) and docosahexaenoic

Chemopreventive effect of omega-3

polyunsaturated fatty acids and

atorvastatin in rats with bladder cancer

Nahla E El-Ashmawy, Eman G Khedr,

Hoda A El-Bahrawy and Samar M Al-Tantawy

Abstract

Bladder cancer remains a huge concern for the medical community because of its incidence and prevalence rates, as well as high percentage of recurrence and progression Omega-3 polyunsaturated fatty acids and atorvastatin proved anti-inflammatory effects through peroxisome proliferator-activated receptor gamma mechanism However, their chemopreventive effect still remained to be examined and clarified In this study, bladder cancer was induced in rats by the chemical carcinogen N-butyl-N-(4-hydroxybutyl)nitrosamine Omega-3 polyunsaturated fatty acids (docosahexaenoic acid and eicosapentaenoic acid: 2:3 w/w; 1200 mg/kg) and/or atorvastatin (6 mg/kg) were given orally daily to rats for eight consecutive weeks concomitantly with N-butyl-N-(4-hydroxybutyl)nitrosamine and continued for further 4 weeks after cessation of N-butyl-N-(4-hydroxybutyl)nitrosamine administration The histopathological examination of rat bladder revealed the presence of tumors and the absence of apoptotic bodies in sections from N-butyl-N-(4-hydroxybutyl) nitrosamine group, while tumors were absent and apoptotic bodies were clearly observed in sections from rat groups treated with omega-3 polyunsaturated fatty acids, atorvastatin, or both drugs The study of the molecular mechanisms illustrated downregulation of COX-2 and P53 (mutant) genes and suppression of transforming growth factor beta-1 and the lipid peroxidation product malondialdehyde in serum of rats of the three treated groups This chemopreventive effect was confirmed by and associated with lower level of bladder tumor antigen in urine However, the combined treatment with both drugs exhibited the major protective effect and nearly corrected the dyslipidemia that has been induced by N-butyl-N-(4-hydroxybutyl)nitrosamine Collectively, omega-3 polyunsaturated fatty acids and atorvastatin, besides having anti-inflammatory properties, proved a chemopreventive effect against bladder cancer, which nominates them to be used as adjuvant therapy with other chemotherapeutics

Keywords

Omega-3 polyunsaturated fatty acids, atorvastatin, N-butyl-N-(4-hydroxybutyl)nitrosamine, bladder cancer,

cyclooxygenase-2, P53

Date received: 1 July 2016; accepted: 9 August 2016

Department of Biochemistry, Faculty of Pharmacy, Tanta University, Tanta, Egypt

Corresponding author:

Samar M Al-Tantawy, Department of Biochemistry, Faculty of Pharmacy, Tanta University, Tanta, 35514 Mansoura, Egypt.

Email: dr.samar680@yahoo.com

Original Article

acid (DHA), are essential very long-chain fatty acids that

contribute to either achieving optimal health or protection

against many diseases, including cancer.4 Several studies

showed chemopreventive effect of EPA and DHA against

several types of cancer such as colorectal cancer,

pancre-atic cancer, and breast cancer.5–7 Their anti-cancer

activi-ties could be attributed to their effect on multiple targets

implicated in different stages of cancer development,

including cell proliferation, angiogenesis, inflammation,

and metastasis.8

Statins are potent inhibitors of cholesterol biosynthesis

and they have showed a great efficacy in decreasing

mor-bidity and mortality from coronary artery disease.9 They

exert their action through inhibition of

3-hydroxy-3-meth-ylglutaryl coenzyme-A (HMG-CoA) reductase, the major

rate-limiting enzyme in the mevalonate pathway of

choles-terol synthesis.10 The inhibition of mevalonate pathway by

statins and its downstream products which are important in

cell signaling, protein synthesis, and cell cycle progression

make statins exhibit many other biological activities,

known as pleiotropic effects Statins have shown

anti-inflammatory, anti-proliferative, antioxidant, and

proapop-totic properties, which could have an important role in

cancer prevention.11

Our target in this work was to investigate the possible

chemopreventive role of n-3 PUFAs and atorvastatin

(ATOR) in bladder cancer and to elucidate the impact of

combined treatment with both drugs on interruption of

some molecular mechanisms involved in cancer

progres-sion, with emphasis on oxidative stress, proliferation, and

apoptosis

Materials and methods

Experimental design

The study was performed in accordance with the

guide-lines for the care and use of laboratory animals approved

by Research Ethics Committee (Faculty of Pharmacy,

Tanta University, Egypt) Male albino rats were utilized in

the study, 120–150 g each Rats were purchased from

National Research Center, Dokki, Giza, Egypt Rats were

weighed and housed in aluminum cages for 2 weeks under

identical environmental conditions and allowed free access

to standard pellet diet and water ad libitum

After acclimatization period, rats were weighed and

randomly divided into five groups: Group 1 (normal control

group; n = 6)—rats in this group were given the vehicle for

12 weeks Group 2

(N-butyl-N-(4-hydroxybutyl)nitrosa-mine (BBN) group; n = 10)—rats in this group received

150 mg/rat orally of BBN (Sigma-Aldrich, Inc, Tokyo,

Japan) dissolved in ethanol:water (1:3 v/v) twice weekly for

8 weeks according to Prasain et al.12 and vehicle for 4 weeks

after the last dose Group 3 (n-3 PUFAs group; n = 8)—rats

in this group received oral dose of 1200 mg/kg/day of n-3

PUFAs (eBioChem, Shanghai, China) for 8 weeks con-comitant with BBN and continued for 4 weeks after cessa-tion of BBN n-3 PUFAs contained DHA and EPA in a ratio of 2:3 w/w dissolved in orange juice Group 4 (ATOR group; n = 8)—rats in this group received oral dose of

6 mg/kg/day of ATOR (Pfizer Inc, New York, NY, USA) for 8 weeks concomitant with BBN and continued for 4 weeks after the cessation of BBN Group 5 (co-treatment group; n = 8)—rats in this group received both n-3 PUFAs and ATOR as in groups 3 and 4

At the end of the experiment (12 weeks), rats were weighed, left in metabolic cages for 12 h for urine collec-tion, and then anesthetized by halothane (Delta Pharma, Heliopolis, Cairo, Egypt) Blood samples were immediately collected by vein puncture from the inferior vena cava and serum was separated Then, the rats were sacrificed by cer-vical dislocation and bladders were dissected Each bladder was washed twice with saline and carefully opened The lumen was blindly inspected by a pathologist for grossly visible lesions, and the number of tumors per rat was cal-culated The length and the width of each tumor were measured by using a caliber, and then the tumor volume was calculated using the following equation: tumor volume = (width2 × length)/2.13 The bladder was then divided into two portions: one portion was preserved in 10% forma-lin for histopathological examination and the other portion was immediately frozen and stored in liquid nitrogen

Histopathological examination

Bladder sections were prepared (3−5 µm thick), stained with hematoxylin and eosin (H&E), and examined blindly

by a pathologist The slides were viewed and images were recorded using Olympus microscope equipped with spot digital camera and computer program MATLAB software

in Pathology Department, Faculty of Veterinary Medicine, Mansoura University, Mansoura, Egypt

Biochemical analysis of serum

Transforming growth factor beta-1 (TGF-β1) was meas-ured in rat serum by rat TGF-β1 enzyme-linked immuno-sorbent assay (ELISA) kit (Hangzhou Sunlong Biotech Company, Ltd., Mainland Shanghai, China) The concen-tration of TGF-β1 was determined according to manufac-turer procedure and expressed as picogram/milliliter The lipid peroxidation marker malondialdehyde (MDA) was measured according to Yoshioka et al.14 using colorimetric kit (Biodiagnostic, Cairo, Egypt), which depends on the reaction of thiobarbituric acid with MDA in acidic medium

at the temperature of 95°C for 30 min to form thiobarbituric acid reactive product Serum cholesterol and triglycerides were measured according to Savoldi et al.15 and Nagele

et al.,16 respectively, using commercial colorimetric kits (Spinreact, S.A., Girona, Spain) High-density lipoprotein

(HDL)-cholesterol (HDL-C) was measured according to

Austin et al.17 after precipitation of very-low-density

lipoprotein (VLDL)-cholesterol and low-density

lipopro-tein (LDL)-cholesterol (LDL-C) by phosphotungstate in

the presence of magnesium ions LDL-C was calculated

from Friedewald equation as follows: LDL-C = total

cholesterol − HDL-C − triglycerides/5.18

Determination of urinary bladder tumor antigen

Bladder tumor antigen (BTA) was measured in rat urine by

rat BTA ELISA kit (Mlbio Biotechnology Company) The

concentration of BTA was determined according to

manu-facturer procedure and expressed as U/mL urine

Quantitative reverse transcription polymerase

chain reaction analysis for COX-2 and P53

Total RNA was extracted from bladder tissue lysed by

RNeasy Plus Mini Kit (Qiagen, Maryland, North America),

and 0.5 µg of total RNA was converted to complementary

DNA (cDNA) by using Thermo Scientific RevertAid First

Strand cDNA Synthesis Kit as the initial step of two-step

reverse transcription polymerase chain reaction (RT-PCR)

protocol A volume of 1 µL of the cDNA was then used

for quantitative PCR using Thermo Scientific Maxima

SYBER Green QPCR Master Mix (2×) The target gene

Ct values were normalized to the Ct value of the

house-keeping gene, glyceraldehyde-3-phosphate

dehydroge-nase (Rat GAPDH), and expressed as relative copy

number (RCN) Primers used in RT-PCR are presented in

Table 1 The primers of P53 (mutant form) and GAPDH

were prepared according to Ibrahim et al.,19 and those

for COX-2 were prepared according to Zuo et al.20 The

relative content of the gene amplification product was

cal-culated using the 2−ΔΔCt method The real-time PCR

instrument (Thermo Fisher Scientific, Waltham, MA,

USA) was adjusted according to the program: 90°C for

1 min (pre-denaturation), and then, 35 cycles (95°C for

30 s, 60°C for 30 s, and 60°C for 30 s)

Statistical analysis

Analysis of data was performed with Statistical Package

for Social Science (SPSS) software version 20.0 All data

are presented as mean ± standard error of the mean (SEM)

Statistical comparison among groups was performed by

one-way analysis of variance (ANOVA) Statistical sig-nificance was obtained at p < 0.05

Results

Survival rate

The survival rate was 60% in BBN group and 75% in treated groups compared to 100% in the normal control group (Figure 1) The increased mortality rate in treated animals may be due to the elevated temperature and long experimental period (12 weeks)

Macroscopic and microscopic examination

The results of macroscopic and microscopic examination are presented in Table 2 and Figures 2–8 In the BBN group, all survived rats (n = 6) had preneoplastic lesions, including hyperplasia (100%), dysplasia (100%), and metaplasia (66.7%) (Table 2), and 83% of the rats had grossly visible bladder tumors (Figure 2(b)) The bladder walls were thicker than those of the control rats, with new

or enlarged small vessels In total, 11 tumors were detected

in five rats (with a mean of tumors 2.2 ± 0.37 per rat) In these five rats, the mean tumor volume was 4.29 ± 0.48 mm3

and the mean tumor volume per tumor was 3.6 ± 1.23 mm3

(Table 2) Histopathological evaluation of BBN group revealed that there were malignant lesions, including car-cinoma in situ (CIS), urothelial carcar-cinoma, adenocarci-noma, and squamous cell carcinoma in addition to the preneoplastic lesions (Table 2, Figure 5) All bladders from the control animals were normal (Figure 2(a)), with trans-lucent and tiny bladders and without any abnormal mass or vascularization Histopathologically, rats of the control

Table 1 Primers for the studied genes.

Figure 1 Survival rate in the studied groups.

Table 2 Macroscopy and microscopy examination results.

Macroscopy (quantitative) and

Tumor number

Tumor volume

Preneoplastic lesions (% (n/n))

Neoplastic lesions (% (n/n))

BBN: N-butyl-N-(4-hydroxybutyl)nitrosamine; SEM: standard error of the mean.

Figure 2 Gross urinary bladder (a) Normal bladder showing normal color (arrow) and normal smooth surface of bladder mucosa

with normal consistency (b) Bladder of BBN group showing intense hemorrhagic and ulcerated mucosa (arrow) with severe thickening in consistency.

Figure 3 Preneoplastic lesions in various treated groups (a) Hyperplasia (arrow): whitish color of bladder mucosa with the

presence of mild nodularity on the surface which is hard in consistency (b) Marked hyperplasia (arrow): bladder mucosa is slightly red in color with nodularity on the surface (c) Dysplasia (arrow): bladder mucosa showing hardness in consistency, abnormal red colored foci, and nodularity on the surface.

group showed normal urothelium with no preneoplastic

lesions (Table 2, Figure 4) The n-3 PUFAs group, ATOR

group, and co-treatment group showed no abnormal

masses or tumors, but some of them showed thicker wall

than normal and preneoplastic lesions mainly hyperplasia

with a ratio of 50%, 33.3%, and 16.7%, respectively (Table

2, Figure 3) The histopathological examination of rats

treated with n-3 PUFAs and/or ATOR concomitant with

BBN showed no neoplastic lesions in bladders but few

preneoplastic lesions were found in some rats The treated rats showed the presence of apoptotic bodies which were not observed in the BBN group (Table 2, Figures 6–8)

Serum transforming growth factor-β1 (TGF-β1)

In the BBN group, there was a significant increase (701 ± 25.4 pg/mL) in serum TGF-β1 (p < 0.05), compared with normal control group (445 ± 13.5 pg/mL) (Figure 9)

Figure 4 Sections of urinary bladder from normal control group showing normal urothelium lining bladder with normal

submucosa (H&E, 100×).

Figure 5 Sections of urinary bladder from BBN group (a) Carcinoma in situ where neoplastic cells are showing marked dysplastic

and neoplastic alterations with tendency to forming cell nests (arrow) without invasion underlying lamina propria and marked esinophilic infiltrate in the lamina propria (H&E, 100×) (b) Squamous cell carcinoma where neoplastic cells have large nucleus, vacuolated with prominent esinophilic nucleoli forming characteristic cell nests (arrow), and has keratin pearl in the center (H&E, 400×) (c) Adenocarcinoma where small densely basophilic neoplastic cells forming acini (arrow) and papillary projections into the lumen of the acini (H&E, 400×) (d) Invasive-type squamous cell carcinoma where nests of neoplastic cells with criteria of malignancy and the presence of the characteristic keratin pearl (arrow) invade the submucosa and muscle layer (H&E, 100×).

Treated groups showed a significant reduction in TGF-β1

(p < 0.001) when compared with the BBN group In n-3

PUFAs, the serum level of TGF-β1 was reduced to

548 ± 14.9 pg/mL; in ATOR group, the serum level was

565 ± 6.3 pg/mL; and in co-treatment group, the serum

level was 518 ± 25.7 pg/mL (Figure 9)

Lipid peroxidation

Serum MDA concentration showed a significant high values

(p < 0.05) in the BBN group (10.4 ± 1.62 nmol/mL) when

compared with control rats (3.7 ± 0.5 nmol/mL) Treated groups showed a significant decrease (p < 0.001) in serum MDA compared with the BBN group The serum level of MDA in n-3 PUFAs group was reduced to 2.8 ± 0.46 nmol/

mL, in ATOR group was 3.6 ± 0.51 nmol/mL, and in co-treatment group was 3.6 ± 0.22 nmol/mL (Figure 10)

Lipid profile

The lipid profile showed increased level of total choles-terol and LDL-C in BBN group compared with normal

Figure 6 Sections of urinary bladder from n-3 PUFAs group (a) Simple hyperplasia of epithelium (arrow) lining urinary bladder

(H&E, 400×) (B) Apoptotic body (arrow) present in the epithelium lining urinary bladder (H&E, 400×).

Figure 7 Sections of urinary bladder from atorvastatin group (a) Dysplastic alteration (arrow) of transitional epithelium lining

bladder (H&E, 100×) (b) Simple hyperplasia (arrow) of lining epithelium of bladder (H&E, 100×).

Figure 8 Sections of urinary bladder from co-treatment group (a) Squamous metaplasia (arrow) of transitional epithelium lining

urinary bladder (H&E, 400×) (b) Apoptosis of hyperplastic cells (arrow) of transitional epithelium lining urinary bladder (H&E, 400×).

control group ATOR treated group and co-treatment

group showed a significant increase in HDL-C and

decrease in LDL-C compared with each of BBN, n-3

PUFAs, and normal control groups n-3 PUFAs treated

group and co-treatment group showed a significant

decrease in triglycerides’ level versus the BBN group

(Table 3)

BTA

In the BBN group, there was a significant increase (84.5 ± 5.38 U/mL) in urinary BTA (p < 0.05) compared with normal control group (40 ± 3.72 U/mL) n-3 PUFAs group showed a significant reduction in urinary level of BTA (55.3 ± 5.4 U/mL) than the BBN group (p < 0.001) BTA was also reduced significantly in ATOR group to 64.4 ± 0.84 U/mL (p < 0.01) and in co-treatment group to 60.5 ± 2.8 U/mL (p < 0.001) versus BBN group (Figure 11) Although decreased in treated groups, urinary BTA remained higher than its value in the normal control group

Real-time PCR for COX-2 and P53 (mutant) in bladder tissue

COX-2 gene expression as a marker of inflammation in bladder cancer tissues showed marked increase in BBN group up to 7.71 fold (p < 0.05) compared with the normal control group In the same way, P53 (mutant form) gene expression was upregulated in BBN group to 11.63 fold (p < 0.05) (Table 4) The gene expression of COX-2 and P53 in rats treated with n-3 PUFAs, ATOR, or both drugs was significantly decreased (p < 0.001) when compared with BBN group, with the greatest suppression of P53 gene expression observed in the co-treatment group rather than mono-treatment groups (Table 4)

Discussion

Bladder cancer is the fifth most frequent tumor in men and ninth in women in the United States.21 Due to a high like-lihood of recurrence, effective chemoprevention is a sig-nificant unmet need.22 This work aims to examine the chemopreventive effect of n-3 PUFAs and ATOR in male rats exposed to induction of bladder cancer and to evaluate the anti-proliferative, anti-inflammatory, proapoptotic, and antioxidant properties of both treatments Bladder cancer was induced in rats in this study by BBN, which is metabo-lized by liver cytochrome P450 into the bladder carcino-gen N-butyl-N-(3-carboxy propyl)nitrosamine (BCPN).23

Table 3 Lipid profile in the studied groups.

HDL-C: high-density lipoprotein cholesterol; LDL-C: low-density lipoprotein cholesterol; TGs: triglycerides; BBN: N-butyl-N-(4-hydroxybutyl) nitrosamine; SEM: standard error of the mean; ATOS: atorvastatin.

Data are expressed as mean ± SEM (n = 6 for each group).

a Significant versus normal control.

b Significant versus BBN group.

c Significant versus n-3 PUFAs group.

Figure 9 Serum levels of transforming growth factor β1 in

the studied groups Data are expressed as mean ± SEM (n = 6

for each group).

a: significant versus negative control; b: significant versus BBN group.

Figure 10 Serum levels of malondialdehyde (MDA) in the

studied groups Data are expressed as mean ± SEM (n = 6 for

each group).

a: significant versus negative control; b: significant versus BBN group.

BCPN reaches the urinary bladder through urine and

comes into contact with the urothelium, binding covalently

to cellular macromolecules, specifically DNA, and

initiat-ing the carcinogenic process.24

Our results indicated that BBN administration twice

weekly for 8 weeks induced neoplastic changes in bladder

of rats which was associated with elevated level of the

uri-nary marker BTA The bladder tissues obtained from all

rats of BBN group showed marked dysplasia, hyperplasia,

CIS, urothelial carcinoma, and squamous cell carcinoma

These histopathological changes were greatly corrected by

treatment of rats with n-3 PUFAs (1200 mg/kg/day) and/or

ATOR (6 mg/kg/day) for 12 weeks Regarding the treated

groups, the bladder sections showed no transitional or

squamous cell carcinoma but the presence of few

preneo-plastic lesions The improvement in the histopathological

changes and the cancerous manifestations in the treated

groups, herein, was associated with and confirmed by the

significant decrease in the urinary BTA, the effect which

was highly apparent in the co-treatment group rather than

mono-treatment groups

Our gene expression study supported the

histopatho-logical findings and indicated that the tumorigenic effect

of BBN was associated with a significant increase in gene expression of COX-2 and the mutant form of P53 Vasconcelos-Nobrega et al.25 demonstrated that BBN-induced rodent tumors, particularly murine tumors, have P53 mutations or mutations in genes related to the P53 pathway Mutation in the P53 gene results in the produc-tion of an abnormal and usually dysfuncproduc-tional mutated P53 with prolonged half-life compared to the wild type, and accumulates in the cell nucleus were proposed to confer a growth advantage to tumor cells.26

Prominent COX-2 expression has been described in bladder cancer, including urothelial and squamous cell car-cinomas, and this expression correlates with tumor grade and invasion.27 COX-2 overexpression by BBN has been implicated in enhanced angiogenesis, which plays a role in carcinogenesis, and is correlated with increased production

of vascular growth factors and formation of capillary net-works.28 COX-2 overexpression may also lead to DNA damage that may eventually lead to carcinogenesis.29

Our data showed that the n-3 PUFAs administration to BBN-exposed rats significantly decreased COX-2 and mutant P53 gene expression, thereby supporting the antitu-morigenic effect of n-3 PUFAs Downregulation of P53 by n-3 PUFAs could be explained by the fact that n-3PUFAs decrease the expression of the mutant form of P53 (mtp53) gene and help the mutant form to restore its normal func-tion and thus leading to enhancement of apoptosis in can-cerous cells.30 This effect was evidenced in this study histopathologically by increased number of apoptotic cells

in bladder tissues of n-3 PUFAs treated rats The proapop-totic properties of n-3 PUFAs were previously examined

by Matsuda et al.,31 who found that n-3 PUFAs can modu-late phosphatase and tensin homolog (PTEN), the expres-sion of which in cancer cell lines upregulates the apoptotic signaling by decreasing nuclear factor kappa B (NF-κB) expression and hence induces tumor regression.32

n-3 PUFAs, in this work, downregulated COX-2 gene expression, which could be attributed to the activation of

Table 4 Cyclooxygenase-2 and P53 gene expression in the studied groups.

RCN: relative copy number; BBN: N-butyl-N-(4-hydroxybutyl)nitrosamine; SEM: standard error of the mean; ATOS: atorvastatin.

Data are expressed as mean ± SEM (n = 6 for each group) (−) Indicates percent decrease.

a Significant versus negative control.

b Significant versus BBN group.

c Significant versus n-3 PUFAs group.

d Significant versus ATOR group.

Figure 11 Urinary levels of bladder tumor antigen (U/mL) in

the studied groups Data are expressed as mean ± SEM (n = 6

for each group).

a: significant versus normal control; b: significant versus BBN group.

peroxisome proliferator-activated receptor gamma (PPAR-γ)

as DHA and EPA act as PPAR-γ ligands.33 Moreover, DHA

and EPA act as competitive inhibitors of arachidonate’s

binding to the active site of COX-2, and EPA acts as an

alternative substrate for this enzyme.34 EPA and DHA were

also reported to have anti-inflammatory role as they can

inhibit the production of a range of other inflammatory

pro-teins, including inducible nitric oxide synthase; TNF-α;

IL-1, IL-6, IL-8, and IL-12 in cultured endothelial cells;

monocytes; macrophages; and dendritic cells.35 Thus,

inhi-bition of COX-2 expression by n-3 PUFAs could be one of

the mechanisms underlying their antitumorigenic effect

This work showed that ATOR downregulated P53

(mtp53) gene expression in bladder of rats exposed to

BBN and exhibited proapoptotic properties confirmed by

histopathological results Previous studies showed that

statins alter Murine double minute 2 (Mdm2) expression

and the P53 response to DNA damage, as statins induce

mammalian target of rapamycin (mTOR)-dependent

Mdm2 phosphorylation at Ser166.36 Phosphorylation of

Mdm2 at Ser166 has been shown to activate Mdm2 and

enhance its ubiquitination ligase function and destabilize

P53 and therefore may help P53 to restore its normal

func-tion and induce apoptosis to cancer cells.37 Statins could

also induce apoptosis through inhibition of RhoA activity,

which in turn activates caspase-dependent mitochondrial

pathway in tumor cells.38

It has been demonstrated that suppression of COX-2 is

one of the pathways required for inhibition of tumor

pro-gression.39 Treatment of rats exposed to BBN with ATOR,

herein, caused downregulation of COX-2 gene

expres-sion in bladder tissue The mechanism underlying this

effect is probably related to inhibition of NF-κB activity

secondary to a decrease in isoprenylation of proteins

involved in intracellular signal transduction COX-2 gene

expression is controlled by NF-κB, and it has been

docu-mented that statins can directly decrease this

transcrip-tion factor.40 Our results were in agreement with many

previous studies which reported that statins downregulate

COX-2 expression in human endothelial cells41 and in

different cell types.42

Elevated expression levels of TGF-β1 have been

reported in several types of cancer such as colorectal,

pros-tate, endometrial, pancreatic, and breast cancer.43,44

TGF-β1 has a negative impact on tumor-surrounding cells,

including the host immune cells, and allows tumor cells to

evade from the immune system.45 TGF-β1 may promote

tumor cell invasion and metastasis by increasing fascin-1

expression.46 A close relationship has been reported

between reactive oxygen species (ROS) and TGF-β1 in

many cancers.47 ROS upregulates TGF-β1 secretion in rat

bladder cancer and activates latent TGF-β1 by oxidation

and/or cleavage of latency-associated protein in a complex

with TGF-β1 when secreted from the cells.48 Moreover,

induction of TGF-β1 responsive genes is mediated by ROS

via mitogen-activated protein kinase (MAPK) pathways and Smad pathway.49

Our laboratory data confirmed the relation between TGF-β1 and ROS and showed elevation of serum TGF-β1 together with the presence of the oxidative stress marker MDA in cancer control group A previous study by Oliveira

et al.50 showed that BBN increases the susceptibility of mitochondrial permeability transition pore to induction by calcium and this can be a consequence of increased ROS production Furthermore, when the nitrosamine com-pounds were metabolized by the liver, they stimulate Kupffer cells leading to the generation of ROS and pre-cipitation of oxidative stress.51

The current data also showed that n-3 PUFAs signifi-cantly reduced serum MDA levels which could be corre-lated with their reported antioxidant properties n-3 PUFAs were demonstrated to modulate antioxidant status in dia-betic rats52 and to inhibit lipid peroxidation in liver and kidney of rats intoxicated with lead acetate.53 The antioxi-dant properties may be related to the assembly of n-3 PUFAs in membrane lipids and lipoproteins making the double bonds less available for free radical attack, inhibi-tion of the pro-oxidant enzyme phospholipase A2, and stimulation of antioxidant enzymes.54 Meanwhile, ATOR also reduced the level of MDA, in this work, when com-pared with the cancer control group, the effect which may

be attributed to inhibition of Rac1/NADPH oxidase activ-ity by ATOR that leads to reduced levels of ROS and hence, decreased oxidative stress.55

The observed antioxidant effect of n-3 PUFAs and ATOR, herein, may explain the suppression of TGF-β1 levels in the rat groups treated with n-3 PUFAs and/or ATOR Moreover, the inhibitory effect of ATOR on the serum levels of TGF-β1 could be attributed to the inhibi-tory effect of ATOR on Smad pathway and also through the inhibition of MAPK/extracellular signal-regulated kinase (ERK) signaling cascade, which is the major path-way controlling cellular processes including growth prolif-eration and survival.56

Regarding lipid profile, the BBN group showed signifi-cantly elevated serum level of total cholesterol, LDL-C, and TGs when compared with normal control group The BBN-induced dyslipidemia was corrected by the co-treat-ment with ATOR and n-3 PUFAs The effects of statins on lipid profile are through the competitive, reversible inhibi-tion of HMG-CoA reductase, the rate-limiting step in cho-lesterol biosynthesis.57 The resultant reduction in cholesterol concentration within hepatocytes triggers upregulation of LDL-receptor expression, which promotes the uptake of LDL and LDL-precursors from systemic cir-culation.58 Statins also inhibit the synthesis of apolipopro-tein B100 and reduce the synthesis and secretion of triglyceride-rich lipoproteins.59 Our findings were in line and showed that ATOR treatment produced a significant reduction in serum total cholesterol and LDL-C and

significantly increased the serum HDL-C when compared

with the BBN group

The current results showed that n-3 PUFAs

signifi-cantly decreased the serum level of triglycerides The

effect of n-3 PUFAs on triglyceride metabolism primarily

includes the suppression of hepatic VLDL synthesis and

discharge with enhancement of VLDL metabolism.60 Since

VLDL is metabolized into intermediate-density

lipopro-tein (IDL) and then LDL, the increased metabolic rate of

VLDL by n-3 PUFAs may account for the increased level

of LDL-C in rats treated with n-3 PUFAs as observed by

our data The beneficial effect of n-3PUFAs

mono-treat-ment in reducing serum triglyceride levels was associated

with elevation of the cardiovascular risk factor LDL-C

However, the combined treatment with both n-3 PUFAs

and ATOR produced a protective effect where all total

cho-lesterol, LDL-C, and TGs were significantly lowered,

whereas HDL-C was significantly elevated

Conclusion

n-3 PUFAs and ATOR, at the studied doses, proved

anti-tum-origenic effect and prevented bladder cancer progression,

which could be through anti-proliferative, anti-inflammatory,

antioxidant, and proapoptotic mechanisms Combined

treat-ment was more effective than either drug alone in

interrupt-ing some molecular targets involved in tumor growth Beinterrupt-ing

of different pharmacological and chemical classes, n-3

PUFAs and ATOR could augment each other, exhibiting high

therapeutic value Further investigations are required to

examine the anticancer effect of the combined treatment

with n-3 PUFAs and ATOR in other cancer models

Acknowledgements

The authors gratefully acknowledge Dr Mohamed Fawzey,

Professor of Histochemistry and Cell Biology, Pathology

Department, Faculty of Veterinary Medicine, Mansoura

University, Mansoura, for conducting and interpreting the

histo-pathological examination.

Declaration of conflicting interests

The author(s) declared no potential conflicts of interest with

respect to the research, authorship, and/or publication of this

article.

Funding

The author(s) received no financial support for the research,

authorship, and/or publication of this article.

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